JP4831711B1 - Anti-Candida fungi and prophylactic and / or therapeutic agents for candidiasis comprising Tamogitake extract as an active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To promote the production of various human β-defensins in humans, which has excellent antibacterial activity such as having fungus growth inhibitory effect and is derived from Tamogitake, an edible mushroom, which is highly safe Β-defensin production promoters, and candidiasis preventive and / or therapeutic agents are provided.
One or more substances selected from the group consisting of an aqueous solvent extract of Tamogitake, glucosylceramide extracted from Tamogitake, ergothioneine extracted from Tamogitake, and ergosterol peroxide extracted from Tamogitake are effective. Ingredients.
[Selection] Figure 1

Description

  TECHNICAL FIELD The present invention relates to an antibacterial agent, β-defensin production promoter and candidiasis preventive and / or therapeutic agent containing Tamimogi extract as an active ingredient, and more particularly, an aqueous solvent extract of Tamimogi, glucosylceramide extracted from Tamomogi The present invention relates to an antibacterial agent, β-defensin production promoter, and candidiasis preventive and / or therapeutic agent, which contains at least one of ergothioneine extracted from Tamogitake and ergosterol peroxide extracted from Tamogitake.

  Tamogittake (Pleurotus cornucopiae var. Citrinopireatus) is a mushroom belonging to the genus Oleander. Tamogitake grows naturally in Hokkaido and Tohoku and is used for food. In recent years, artificially grown products are also sold.

  Tamogitake is known to have various components useful in the food industry, health industry, cosmetics industry, pharmaceutical industry, etc. For example, Patent Document 1 discloses an antibacterial agent containing a water extract of Tamogittake as an active ingredient. Patent Document 2 and Patent Document 3 disclose an antitumor immunostimulant containing a hot water extract of Tamogitake as an active ingredient, a dermatitis therapeutic agent and a skin moisturizer containing a ceramide derived from Tamogitake as active ingredients. Patent Document 4 discloses an antioxidant, tyrosinase activity inhibitor, lipase activity inhibitor, 5α-reductase activity inhibitor, anti-aging agent, and anti-metabolic syndrome agent containing ergothioneine derived from Tamogitake as an active ingredient. Is an anti-discoloration and anti-oxidant for foods containing ergosterol peroxide from Tamogitake as an active ingredient It is shown.

JP 2007-1961 International Publication WO2006 / 038527 Pamphlet JP 2007-308394 A JP 2009-126863 A JP 2009-183266 A

  However, the antibacterial agent containing the water extract of Tamogitake disclosed in Patent Document 1 as an active ingredient has only been shown to suppress the growth of the fungus in the culture medium. No suppression has been shown. In addition, the Tamogitake hot water extract, the ceramide derived from Tamogitake, the ergothioneine derived from Tamogitake and the ergosterol peroxide derived from Tamogitake described in Patent Documents 2 to 5, are all antibacterial agents, β-defensin production promoters, and candidiasis It is not used as a preventive and / or therapeutic agent.

  The present invention has been made in order to solve the above-mentioned problems, has an excellent antibacterial activity such as having an effect of inhibiting the growth of bacteria, and is high because it is derived from Tamogitake, an edible mushroom. It is an object to provide an antibacterial agent having safety, a β-defensin production promoter capable of promoting the production of various human β-defensins in humans, and an agent for preventing and / or treating candidiasis.

  As a result of intensive studies, the present inventors have found that a hot water extract of Tamogitake inhibits the growth of Candida fungi in the culture medium and the oral cavity of an oral candidiasis model mouse, and an aqueous solvent extract of Tamogitake, extracted from Tamogitake It was found that glucosylceramide, ergothioneine extracted from Tamogitake, and ergosterol peroxide extracted from Tamogitake promote the production of β-defensin, and the following inventions were completed.

(1) One or more substances selected from the group consisting of an aqueous solvent extract of Tamogitake, glucosylceramide extracted from Tamogitake, ergothioneine extracted from Tamogitake, and ergosterol peroxide extracted from Tamogitake Antibacterial agent.

(2) The antibacterial agent according to (1), wherein the aqueous solvent extract is a hot water extract.

(3) One or more substances selected from the group consisting of an aqueous solvent extract of Tamogitake, glucosylceramide extracted from Tamogitake, ergothioneine extracted from Tamogitake, and ergosterol peroxide extracted from Tamogitake Β-defensin production promoter.

(4) β-defensin production promotion according to (3), wherein β-defensin is one or more β-defensins selected from the group consisting of human β-defensin-1, human β-defensin-2 and human β-defensin-3 Agent.

(5) The β-defensin production promoter according to (3) or (4), wherein the aqueous solvent extract is a hot water extract.

(6) One or more substances selected from the group consisting of an aqueous solvent extract of Tamogitake, glucosylceramide extracted from Tamogitake, ergothioneine extracted from Tamogitake, and ergosterol peroxide extracted from Tamogitake An agent for preventing and / or treating candidiasis.

(7) The candidiasis preventive and / or therapeutic agent according to (6), which is used for a patient with xerostomia.

(8) The agent for preventing and / or treating candidiasis according to (6) or (7), wherein the aqueous solvent extract is a hot water extract.

  According to the present invention, an antibacterial agent having excellent antibacterial activity such as having an effect of inhibiting the growth of bacteria and being derived from Tamogitake, which is an edible mushroom, has high safety. It is possible to obtain a β-defensin production promoter capable of promoting production, and an agent for preventing and / or treating candidiasis.

It is a figure which shows the result of having measured the turbidity of the culture medium after culture | cultivating Candida in the culture medium which added the Tamogitake hot-water extract. The vertical axis represents the turbidity (absorbance at 600 nm), and the horizontal axis represents each sample in which the addition concentration of the Tamogitake hot water extract was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 24 hours by the culture medium which added the Tamogitake hot-water extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of the Tamogitake hot water extract was changed. It is a figure which shows the mRNA relative expression level of hBD-3 in the HaCaT cell culture | cultivated for 24 hours by the culture medium which added the filtrate, filtrate, and Tamogitake hot water extract of the Tamogitake hot water extract. The vertical axis shows the relative expression level of mRNA, and the horizontal axis shows each sample obtained by adding the filtrate, filtrate and Tamogitake hot water extract to the exchange medium. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 24 hours by the culture medium which added the bamboo grass water extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of the water extract of Tamogitake was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 24 hours by the culture medium which added the bamboo shoot acidic aqueous solvent extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of Tamogitake acidic aqueous solvent extract was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 24 hours by the culture medium which added the Tamogitake basic aqueous solvent extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of Tamogitake basic aqueous solvent extract was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 24 hours by the culture medium which added the bamboo shoot derived ergothioneine. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of ergothioneine derived from Tamogitake was varied. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 24 hours by the culture medium which added the bamboo shoot derived ergosterol peroxide. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the concentration of added ergosterol peroxide was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell cultured for 24 hours by the culture medium which added the glucosylceramide derived from Tamogitake. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of glutinous glucosylceramide was changed. It is a figure which shows the mRNA relative expression level of hBD-2 and hBD-3 in the NHEK cell culture | cultivated for 24 hours by the culture medium which added the Tamogitake hot-water extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of the Tamogitake hot water extract was changed. It is a figure which shows the mRNA relative expression level of hBD-2 and hBD-3 in the HaCaT cell culture | cultivated for 2 hours by the culture medium which added the bamboo grass hot water extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of the Tamogitake hot water extract was changed. It is a figure which shows the mRNA relative expression level of hBD-3 in the HaCaT cell culture | cultivated for 2 hours by the culture medium which added the filtrate, filtrate, and Tamogitake hot water extract of the Tamogitake hot water extract. The vertical axis shows the relative expression level of mRNA, and the horizontal axis shows each sample obtained by adding the filtrate, filtrate and Tamogitake hot water extract to the exchange medium. It is a figure which shows the mRNA relative expression level of hBD-3 in the HaCaT cell culture | cultivated for 2 hours by the culture medium which added the ultrasonic treatment thing of the bamboo shoot bamboo extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of the sonicated product of the Tamogitake hot water extract was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 2 hours by the culture medium which added the bamboo grass water extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of the water extract of Tamogitake was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 2 hours by the culture medium which added the bamboo shoot acidic aqueous solvent extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of Tamogitake acidic aqueous solvent extract was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 2 hours by the culture medium which added the Tamogitake basic aqueous solvent extract. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of Tamogitake basic aqueous solvent extract was changed. It is a figure which shows the mRNA relative expression level of hBD-1, hBD-2, and hBD-3 in the HaCaT cell culture | cultivated for 2 hours by the culture medium which added the tarogotake ergothioneine. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents each sample in which the addition concentration of ergothioneine derived from Tamogitake was varied. The figure which shows the relative expression level of mRNA of hBD-2 and hBD-3 in the NHEK cell culture | cultivated for 2 hours by the culture medium which added the Tamogitake hot-water extract with Suehiro bamboo origin beta glucan or mycoplasma origin diacyl lipopeptide (FSL-1). is there. The vertical axis represents the relative expression level of mRNA, and the horizontal axis represents Suehirotake-derived β-glucan, barley-derived β-glucan, FSL-1, Tamogitake hot water extract, Tamogitake hot-water extract + Suehirotake-derived β-glucan, FSL-1 + Each sample to which Suehirotake-derived β-glucan and Tamogitake hot water extract + FSL-1 were added is shown. It is a graph which shows the viable count of Candida bacteria in the tongue of the oral candidiasis model mouse which administered the gel for oral cavity containing a Tamogitake hot water extract. The vertical axis represents the viable count of Candida, and the horizontal axis represents the group that did not administer the oral gel (control group) and the group that administered the oral gel (gel administration group).

  Hereinafter, the antibacterial agent, β-defensin production promoter and candidiasis preventive and / or therapeutic agent according to the present invention will be described in detail. The antibacterial agent according to the present invention is one or more selected from the group consisting of an aqueous solvent extract of Tamogitake, glucosylceramide extracted from Tamogitake, ergothioneine extracted from Tamogitake, and ergosterol peroxide extracted from Tamogitake These substances are the active ingredients.

  In the present invention, Tamogitake may be naturally grown or artificially cultivated. Moreover, the raw bamboo shoots may be sufficient and what was processed after extract | collecting, such as what was dried and what was made into powder after drying, may be used.

  In the present invention, the aqueous solvent extract of Tamogitake is a liquid, semi-solid or solid substance containing a component extracted from Tamogitake with an aqueous solvent, or one or more selected from these substances A mixture of substances. Here, in the present invention, the aqueous solvent is not particularly limited as long as it is a solvent mainly composed of a polar solvent, and may be composed of one type of solvent or a mixture of two or more types of solvents. Examples of such a polar solvent include water, methanol, ethanol, acetic acid, formic acid, 1-butanol, 1-propanol, 2-propanol and the like, and water is preferable.

  In the present invention, the aqueous solvent may be prepared by mixing or dissolving an appropriate solute in advance in water or an aqueous solvent to form an aqueous solvent. Examples of such an aqueous solvent include mixing or dissolving citric acid in water. In addition, a mixture of sodium bicarbonate in or dissolved in water, a mixture of sodium chloride in acetic acid or the like, and citric acid aqueous solution, sodium bicarbonate aqueous solution and sodium chloride acetic acid solution are preferable.

  In the present invention, the aqueous solvent may be acidic, neutral or basic, and the temperature of the aqueous solvent is not particularly limited as long as the function is not impaired. Good. In the present invention, hot water can be used as a suitable aqueous solvent.

  In the present invention, an aqueous solvent extract of Tamogitake can be produced according to a conventional method. For example, Tamogitake is added to an aqueous solvent, heated, ground or stirred, and then solid-liquid separated by filtration or the like to recover liquid components. Can be manufactured. The produced aqueous solvent extract of Tamogitake may be further subjected to ultrafiltration, sonication, etc., and such ultrafiltrate (filtrate and filtrate), sonication, etc. are also treated with Tamogitake. And is included in the present invention.

  In the present invention, glucosylceramide extracted from bamboo shoots can be produced according to known or new techniques that can be appropriately selected by those skilled in the art. For example, boiled bamboo shoots are dried and pulverized into powder. The resulting extract is saponified with an aqueous NaOH solution, adjusted to pH with dilute hydrochloric acid, centrifuged, and the supernatant is removed to add ethanol to the precipitate. And a method of repeatedly purifying the precipitate by column chromatography using a silica gel column and a chloroform / methanol solution.

  In the present invention, ergothioneine extracted from bamboo shoots can also be produced according to a known or new technique that can be appropriately selected by those skilled in the art. For example, boiled juice obtained by boiling bamboo shoots was used as an ion exchange resin. Thereafter, a method may be mentioned in which a cationic compound is eluted from the resin, the eluate is concentrated, separated and purified by high performance liquid chromatography, and lyophilized.

  In the present invention, ergotisterol peroxide extracted from bamboo shoots can also be produced according to known or new techniques that can be appropriately selected by those skilled in the art, for example, the same as the above-described method for producing glucosylceramide. And a method of selecting an eluate containing ergosterol peroxide instead of glucosylceramide at the time of purification by column chromatography.

  In the present invention, in the production of an aqueous solvent extract of Tamogitake, glucosylceramide extracted from Tamogitake, ergothioneine extracted from Tamogitake and ergosterol peroxide extracted from Tamogitake, the purity and shape required Depending on the situation, pulverization, purification, concentration, drying, sterilization and the like may be performed.

  As the pulverization method, for example, a method of crushing with a roll type pulverizer, a method of cutting with a food processor, a method of crushing with a ball mill pulverizer, a method of pulverizing with a hammer type pulverizer, etc. Examples of the method include a filtration method, a distillation method, a recrystallization method, a reprecipitation method, and a method using various chromatographies. Examples of the concentration method include vacuum concentration (vacuum concentration using a boiling concentration method, an evaporator, etc.). ) Method, freeze concentration method, membrane concentration method using reverse osmosis membrane and the like, and examples of sterilization methods include a method using an autoclave, a high frequency method, a filter filtration method and the like.

  The antibacterial agent according to the present invention has an action of suppressing the growth of microorganisms. In the present invention, “suppressing the growth of microorganisms” refers to the case of suppressing the growth by suppressing the division of microorganisms. In addition, the case where growth is suppressed by killing microorganisms is included.

  In addition to the action of suppressing the growth of microorganisms by acting directly on microorganisms (direct action), the action of inhibiting the growth of microorganisms is also the action of antimicrobial peptides by acting on the antimicrobial peptide-producing cells in the living body. The action (indirect action) of promoting production and inhibiting the growth of microorganisms by the produced antimicrobial peptide is included.

  In the present invention, “suppressing proliferation” means “inhibiting proliferation”, “blocking proliferation”, “preventing proliferation”, and “promoting production” means “increasing production”, It is used interchangeably with “enhance production”, “enhance production”, “enhance production”, “stimulate production”, and “activate production ability”.

  Antibacterial peptide is a generic term for peptides that are produced in cells of a wide range of organisms, from microorganisms to animals and plants including humans, and have bacteriostatic effects such as killing action on microorganisms, suppression or inhibition of division, etc. . In the present invention, the antibacterial peptide is not particularly limited, and examples thereof include α-defensin, β-defensin, θ-defensin, Catericidin, Darmcidin, Hepcidin / LEAP-1, and histatin.

  That is, the antibacterial agent according to the present invention is effective for inhibiting the growth of microorganisms and preventing or treating diseases caused by microorganisms. For example, the antibacterial agents are added to pharmaceuticals and quasi-drugs for animals including humans. In addition, it can be used as an agrochemical that prevents plant diseases such as crops. Examples of microorganisms include not only bacteria and fungi but also all microorganisms such as viruses, viroids, rickettsia, chlamydia, mycoplasma, spirochetes, protozoa, and helminths. Examples of diseases caused by such microorganisms include infectious diseases caused by microorganism infections, poisoning caused by toxins produced by microorganisms, and allergic diseases, such as candidiasis, cryptococcus infections, aspergillosis, Pneumocystis pneumonia, caries, food poisoning, HIV infection, ringworm (athlete's foot, worm, shirakumo, etc.), flying fish, chickenpox, herpes virus infection, cytomegalovirus infection, MRSA infection, Pseudomonas aeruginosa infection, Legionella pneumonia , Serratia infection, toxoplasmosis, cryptosporidiosis and the like.

  The antibacterial agent according to the present invention can also be added to foods and cosmetics and used to enhance its preservability, and is added to and blended with articles to prevent corrosion, thus maintaining a hygienic state. Or it can be used to prevent malodors.

  Next, the present invention provides a β-defensin production promoter. The β-defensin production promoter according to the present invention is selected from the group consisting of an aqueous solvent extract of Tamogitake, glucosylceramide extracted from Tamogitake, ergothioneine extracted from Tamogitake, and ergosterol peroxide extracted from Tamogitake Alternatively, two or more substances are used as active ingredients.

  Defensin, a kind of antibacterial peptide, is one of the family of antibacterial peptides characterized by having a cysteine residue in the molecule and forming a higher order structure by a disulfide bond. It is roughly divided into defensin and θ-defensin. Since defensin is positively charged, it is known that it is inserted into a negatively charged cell membrane to form a small pore, and the antibacterial action is exhibited by the outflow of the cell contents from the small pore. .

  β-Defensin is a defensin produced mainly from leukocytes and epithelial cells in vivo. In recent years, not only local infection protection but also tissue repair of epithelial tissues and migration of dendritic cells, T lymphocytes, monocytes, etc. (Yang D. et al., Science, 286, 525, 1999; Territo MC et al., J. Clin. Invest. 84, 2017, 1989; Lillard JW Jr. et al., Proc. Natl. Acad. Sci., 96, 651, 1999; Isao Nagaoka et al., Clinical Immunity, 33 577, 2000). It has also been reported that tumor immunity is induced to exert an anti-tumor effect and that cancer cells have a growth-suppressing effect (Isao Nagaoka et al., Clinical Immunity, Vol. 40, No. 4, 424) 2003; Byragyn A et al., J. Immunol., 167, 6644, 2001).

  That is, the β-defensin production promoter according to the present invention is effective for the prevention and treatment of diseases caused by microorganisms. For example, it is used by adding and blending drugs and quasi drugs with animals including humans. In addition, it can be used for pesticides that prevent plant diseases such as crops. Further, the β-defensin production promoter according to the present invention is used for recovery from infection, inflammation and other injuries, cancer treatment, immune enhancement, skin care for rough skin, dry skin, rough hands, prevention of bad breath, foot odor, etc. Can be used.

  In the present invention, examples of the type of β-defensin include β-defensin-1, β-defensin-2, β-defensin-3, β-defensin-4, and the like, and examples of the living organisms include, for example, Lactococcus lactis Bacteria, Ashitaba, Karashina, etc., Fungi, such as Black-bellied bamboo, Insects, such as Spider, Scorpion, Dragonfly, Molluscs, such as mussels, Crustaceans, such as lobsters, Birds, such as chickens, Amphibians, such as frogs, Humans, Monkeys (Primates excluding humans), mammals such as cattle, pigs, sheep, goats, dogs, cats, etc. can be mentioned, but humans are preferred.

  Next, the present invention provides an agent for preventing and / or treating candidiasis. The agent for preventing and / or treating candidiasis according to the present invention is selected from the group consisting of an aqueous solvent extract of Tamogitake, glucosylceramide extracted from Tamogitake, ergothioneine extracted from Tamogitake, and ergosterol peroxide extracted from Tamogitake One or two or more substances are used as active ingredients.

  Candidiasis is C.I. It is an infectious disease caused by Candida bacteria represented by albicans. Candida always lives on the human body surface, digestive tract, vaginal mucosa, etc., and has no particular effect on healthy people, but when physical condition is poor or immunity is reduced, It is an opportunistic infection that causes disease. The agent for preventing and / or treating candidiasis according to the present invention acts on the antibacterial peptide-producing cells in addition to the action (direct action) of inhibiting its growth by directly acting on Candida bacteria. Production is promoted, and the produced antimicrobial peptide has an action (indirect action) of suppressing the growth of Candida.

  Examples of candidiasis in the present invention include cutaneous candidiasis, oral candidiasis, genital candidiasis, stomatitis, stomatitis, gastric bloating, candida sepsis, etc. Among these, oral candidiasis is dry mouth It is known to occur frequently in patients with illness (dry mice). Xerostomia (dry mouth) is a disease in which the oral cavity is dry and causes symptoms such as thirst, thirst, pain, bad breath, and abnormal taste. It is considered that Candida bacteria invade the oral epithelium and cause oral candidiasis because the balance of the resident bacteria in the oral cavity is lost. The candidiasis and / or therapeutic agent according to the present invention is particularly effective for such oral candidiasis.

  Methods known to those skilled in the art can be used to formulate the antibacterial agent, β-defensin production promoter and candidiasis preventive and / or therapeutic agent according to the present invention. The dosage form may also be a dosage form that can be appropriately selected by those skilled in the art. Examples of such a dosage form include tablets, granules, powders, capsules, and coating agents in the case of preparation as an oral dosage formulation. In the case of a parenteral preparation, gel, inhalation, injection, infusion, suppository, coating agent, spray, patch, ointment, cream And the like. In addition, the dosage can be appropriately set depending on the formulation form of the pharmaceutical composition, the administration method, the purpose of use, and the age, weight and symptom of the administration subject applied thereto.

  When used as a skin external preparation, the dosage form can be appropriately selected. For example, a solution system, a solubilization system, an emulsification system, a powder dispersion system, a water-oil two-layer system, and a water-oil-powder three-layer system. In addition to systems, gels, mists, sprays, mousses, roll-ons, sticks, and the like, preparations impregnated or coated on sheets such as nonwoven fabrics can be exemplified. Further, the antibacterial agent, β-defensin production promoter and candidiasis prevention and / or treatment agent according to the present invention include, for example, oils and fats such as vegetable oil, waxes such as lanolin and beeswax, hydrocarbons, fatty acids and higher alcohols. , Esters, various surfactants, pigments, fragrances, vitamins, plant and animal extract components, UV absorbers, antioxidants, antiseptics, bactericides, etc. The skin external preparation can be produced by appropriately blending the above. Further, it can be used together with licorice extract components such as glycyrrhetinic acid, which is a raw material for external preparations for anti-inflammatory skin, diphenhydramine hydrochloride, azulene, dl-α-tocopherol and its derivatives, vitamin B2, vitamin B6 and the like.

  When used as a food additive, its form can be selected as appropriate, for example, the antibacterial agent, β-defensin production promoter and candidiasis preventive and / or therapeutic agent according to the present invention prepared as it is as a food, Examples include those added to other foods, or any form usually used for foods or health foods such as capsules and tablets. In addition, when ingested or administered by blending in food, appropriately mixed with excipients, extenders, binders, thickeners, emulsifiers, colorants, fragrances, food additives, seasonings, etc. Depending on the application, it can be formed into a powder, granule, tablet or the like. Furthermore, it can be ingested by preparing a food by mixing it into a food material and commercializing it as a functional food.

  Hereinafter, the antibacterial agent, β-defensin production promoter and candidiasis preventive and / or therapeutic agent according to the present invention will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.

<Example 1> Confirmation of Candida fungus growth inhibitory effect (1) Preparation of Tamogitake hot water extract Raw Tamogitake 150 kg to 200 kg is heated in 200 L of water, boiled for 5 minutes and then filtered. In order to obtain 150 L of the filtrate, a Tamogitake hot water extract was prepared.

(2) Preparation of sample Prepare 4 sheets of brain heart fusion medium (BHI medium; Eiken Chemical Co., Ltd.) in each well of a 6-well plate (IWAKI). Four samples were prepared. Candida (Candida albicans ATCC 90028) was inoculated into each well of each sample so that the absorbance at 600 nm of the culture solution was 0.01 to 0.05. The Tamogitake hot water extract prepared in Example (1) was added to 6% (v / v), 8% (v / v) and 60% (v / v), respectively. In addition, A well to which nothing was added was used as a control.

(3) Candida culturing and measurement of turbidity A, B, C and D of Example (2) were cultured at 25 ° C. for 16 hours. Next, turbidity was determined by measuring the absorbance at a wavelength of 600 nm using a spectrophotometer (BioRad) for the cultured medium as an indicator of the number of viable Candida bacteria. Subsequently, the average value and standard deviation of turbidity in each well were calculated for each sample. In addition, for the calculated results in B, C, and D, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the test is statistically significant (p <0.05).

  As shown in FIG. 1, the magnitude of turbidity is A> B> C> D. When the turbidity in A is 100%, the turbidity in B, C, and D is about 92% and about 82%, respectively. And 58-71%. That is, it was confirmed that the viable count of Candida spp. Was smaller as the concentration of the hot water extract added to the medium was higher.

  From these results, it was clarified that Tamogitake hot water extract suppresses the growth of Candida.

<Example 2> Examination of human β-defensin production promoting effect (1) Examination of human β-defensin production promoting effect of Tamogitake hot water extract [1-1] Sample preparation and culture Fetal bovine serum (FBS; Gibco- BRL) and penicillin streptomycin (Sigma) at final concentrations of 10% (v / v) and 2% (v / v), respectively, were prepared, and FP-DMEM medium and did. A cell suspension was prepared by adding human immortalized keratinocyte cells (HaCaT cells; provided by Takashi Muramatsu, Department of Pathology, Tokyo Dental College) to 1 × 10 ⁵ cells / mL in FP-DMEM medium. . Five pieces of 3 mL of this cell suspension placed in a 60 mm dish (IWAKI) were prepared, and a total of 5 samples were prepared as A, B, C, D and E. Subsequently, each sample was cultured in an environment of 37 ° C. and CO ₂ 5% (v / v) until reaching 80% confluence for about 3 days.

[1-2] Culture in medium supplemented with Tamogitake hot water extract The Tamogitake hot water extract prepared in Example 1 (1) was diluted with DDW and 2% (v / v) each. 20% (v / v) and 40% (v / v) hot water extract aqueous solutions were prepared. Next, DDW, 2% (v / v), 20% (v / v), 40% (v / v) hot water extract aqueous solution and Tamogitake hot water extract prepared in Example 1 (1) were used. It added to FP-DMEM culture medium so that it might become 25% (v / v), respectively, and the exchange medium for control and the exchange medium which contains a Tamogitake hot water extract with the following final concentration were prepared. After the medium of each sample of this example (1) [1-1] was replaced with the prepared replacement medium as described below, under the environment described in this example (1) [1-1], Cultured for 24 hours.

What was added to the exchange medium and the final concentration A of hot water extract of Tamogitake A: DDW (exchange medium for control)
B: Tamogitake hot water extract final concentration 0.5% (v / v)
C: Tamogitake hot water extract final concentration 5% (v / v)
D: Tamogitake hot water extract final concentration 10% (v / v)
E: Tamogitake hot water extract final concentration 25% (v / v)

[1-3] Extraction of RNA and preparation of cDNA After recovering cells from each sample of Example (1) [1-2], total RNA in the cells was extracted using TRIzol reagent (Invitrogen). . Subsequently, 10% (v / v) volume of chloroform of the extracted total RNA was added, and the mixture was centrifuged at 13000 rpm and 4 ° C. for 10 minutes, and then the aqueous layer was recovered. Next, after adding an equal volume of isopropanol and centrifuging for 10 minutes under the conditions of 13000 rpm and 4 ° C., the supernatant was removed, and 600 μL of 70% (v / v) ethanol was added to the precipitate. Was washed. Thereafter, the precipitate was dissolved in sterilized DEPC-treated water to obtain a total RNA solution. An appropriate amount was taken from this total RNA solution, and the concentration was measured by measuring the absorbance at a wavelength of 260 nm using a spectrophotometer NanoDrop ND-1000 (NanoDrop products).

Subsequently, cDNA was prepared using an oligo dT ^12-18 primer (Invitrogen) and SuperScript reverse transcriptase (Invitrogen) as a template using a total RNA solution containing 2 μg of total RNA as a template.

[1-4] Quantification of human β-defensin mRNA by quantitative PCR method Using the cDNA of Example (1) [1-3] as a template, human β-defensin-1 (hBD-1), human β-defensin- 2 (hBD-2), human β-defensin-3 (hBD-3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Taq Man 7300 Real-Time PCR System (Applied Biosystems) and Taq Man Universal Using Master Mix (Applied Biosystems), the quantitative PCR method was performed according to the attached instruction manual, and the number of cycles (Ct value) that resulted in a constant amount of amplified product was measured. Primers and probes used in the quantitative PCR method are as follows.

Primer / probe Taq Man Gene Expression Assays (Applied Biosystems) used for quantitative PCR;
Primer / probe used for amplification of hBD-1: Assay ID Hs00608345_m1
Primer / probe used for amplification of hBD-2: AssayID Hs00823638_m1
Primer / probe used for amplification of hBD-3: AssayID Hs00218678_m1
Primer / probe used for amplification of GAPDH: AssayID Hs99999999_m1

  Subsequently, relative expression levels of hBD-1, hBD-2 and hBD-3 mRNA were obtained by performing a comparative Ct method using the Ct value in A as a reference value and the CPD value of GAPDH as an intrinsic control based on the measurement results. Was calculated.

  In this example (1), the experiment of [1-1] to [1-4] was repeated three times or more, and the average value and the standard for the relative expression levels of hBD-1, hBD-2 and hBD-3 in each sample. Deviation was calculated. In addition, for the calculated results in B, C, and D, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 2, regarding the relative expression level of hBD-1 mRNA, all of B, C, D and E were almost the same as A.

  In addition, as shown in the middle part of FIG. 2, regarding the relative expression level of hBD-2 mRNA, B was hardly changed compared to A, but C and E tended to be larger than A, and D was It was significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-2 was increased when HaCaT cells were cultured in a medium supplemented with Tamogitake hot-water extract, compared with the case where the cells were cultured without adding them.

  In addition, as shown in the lower part of FIG. 2, regarding the relative expression level of hBD-3 mRNA, B and E tended to be larger than A, and C and D were significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-3 was increased when HaCaT cells were cultured in a medium supplemented with an extract of Tamogitake hot water, as compared with the case where the cells were cultured without adding them.

  From these results, it was revealed that the Tamogitake hot water extract promotes the production of hBD-2 and hBD-3 when allowed to act on HaCaT cells for 24 hours.

(2) Examination of human β-defensin production promoting effect of filtrate and filtrate of Tamogitake hot water extract [2-1] Preparation of filtrate and filtrate of Tamogitake hot water extract Tamogitake prepared in Example 1 (1) About 150 mL of hot water extracts, ultrafiltration was performed using an ultrafiltration membrane having a fractional molecular weight of 1000 to obtain 25 mL of filtrate and filtrate (dry weight; 0.2 g).

[2-2] Preparation and Culture of Samples A total of 4 samples A, B, C, and D were prepared and cultured by the method described in Example (1) [1-1].

[2-3] Culture of Tamogitake hot water extract in a filtrate and a culture medium to which the filtrate was added The filtrate of Example (2) [2-1] was dissolved in DDW, and a 1 mg / mL filtrate aqueous solution was dissolved. Prepared. Next, DDW, the filtrate of Example (2) [2-1], the filtrate aqueous solution, and the hot water extract prepared in Example 1 (1) were each 10% (v / v) F- It was added to P-DMEM medium to prepare a control exchange medium and an exchange medium containing a filtrate, a filtrate, and a hot water extract. After replacing the medium of A, B, C and D in Example (2) [2-2] with the prepared exchange medium as described below, the environment described in Example (1) [1-1] The culture was further continued for 24 hours.

Added to the exchange medium A: DDW (exchange medium for control)
B: Filtrate C: Filtrate D: Tamogitake hot water extract

[2-4] Extraction of RNA, preparation of cDNA, and quantification of hBD-3 mRNA by quantitative PCR method For each sample of this Example (2) [2-3], this Example (1) [1-3] After extracting RNA and preparing cDNA by the method described in 1. above, quantitative PCR was performed by the method described in Example (1) [1-4] to quantify hBD-3 mRNA. In addition, for the calculated results in B, C, and D, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in FIG. 3, the relative expression level of hBD-3 mRNA was A <B <C <D, and B, C, and D were significantly larger than A.

  From these results, it is clear that the fraction (filtrate) that passed through the ultrafiltration membrane having a molecular weight cut off of 1000 and the fraction that did not pass through it (filtrate) were both expressed in HaCaT cells. It was revealed that hBD-3 production was promoted when allowed to act for 24 hours.

(3) Examination of human β-defensin production promoting effect of water extract of Tamogitake [3-1] Preparation of water extract of Tamogitake 100 g of raw Tamogitake and 100 g of water were put into a food processor and pulverized. And filtered to give 70 mL of filtrate. It was pH 6.08 when pH of the obtained filtrate was measured. Subsequently, the filtrate was sterilized at 120 ° C. for 15 minutes using an autoclave apparatus.

[3-2] Preparation and culture of sample, and culture in medium supplemented with Tamogitake water extract By the method described in Example (1) [1-1], A, B, C, D and E A total of 5 samples were prepared and cultured. Subsequently, instead of the Tamogitake hot water extract prepared in Example 1 (1), the Tamogitake water extract prepared in this Example (3) [3-1] was used, and this Example (1) [ In the method described in [1-2], culturing was carried out in a medium supplemented with an aqueous extract of Tamogitake.

[3-3] Extraction of RNA, preparation of cDNA, and quantification of human β-defensin mRNA by quantitative PCR method For each sample of Example (3) [3-2], this Example (1) [1- 3] After extracting RNA and preparing cDNA by the method described in [3], quantitative PCR is performed by the method described in Example (1) [1-4] to quantify human β-defensin mRNA. went. In addition, for the calculated results in B, C, D, and E, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 4, regarding the relative expression level of hBD-1 mRNA, B was hardly changed compared to A, but C, D, and E were significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-1 was increased when HaCaT cells were cultured in a medium supplemented with an extract of Tamogitake water, compared to when cultured without adding them.

  Further, as shown in the middle of FIG. 4, regarding the relative expression level of hBD-2 mRNA, all of B, C, D, and E were almost the same as A.

  Further, as shown in the lower part of FIG. 4, regarding the relative expression level of hBD-3 mRNA, B and C were almost the same as A, but D and E were significantly larger than A. . That is, it was confirmed that the mRNA expression level of hBD-3 increased when HaCaT cells were cultured in a medium supplemented with an extract of Tamogitake water, as compared to the case where the cells were cultured without adding them.

  From these results, it was revealed that the water extract of Tamogitake promotes the production of hBD-1 and hBD-3 when allowed to act on HaCaT cells for 24 hours.

(4) Examination of human β-defensin production promoting effect of Tamogitake acidic solvent extract [4-1] Preparation of Tamogitake acidic solvent extract This implementation was carried out by replacing water with 0.4% (w / w) citric acid aqueous solution. Example (3) An extract of Tamogitake was prepared by the method described in [3-1], and this was used as an acidic solvent extract of Tamogitake. In addition, pH of the filtrate before performing sterilization by an autoclave was pH 4.61.

[4-2] Preparation and culture of sample, and culture in medium supplemented with Tamogittake acidic solvent extract A, B, C, D and E according to the method described in Example (1) [1-1] A total of 5 samples were prepared and cultured. Then, it replaced with the Tamogitake hot-water extract prepared in Example 1 (1), and used the Tamogitake acidic solvent extract prepared in this Example (4) [4-1], and this Example (1). By the method as described in [1-2], it culture | cultivated by the culture medium which added the bamboo shoot acidic solvent extract.

[4-3] Extraction of RNA, preparation of cDNA, and quantification of human β-defensin mRNA by quantitative PCR method For each sample of this example (4) [4-2], this example (1) [1- 3] After extracting RNA and preparing cDNA by the method described in [3], quantitative PCR is performed by the method described in Example (1) [1-4] to quantify human β-defensin mRNA. went. In addition, for the calculated results in B, C, D, and E, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 5, regarding the mRNA relative expression level of hBD-1, B, C, and D were hardly changed compared to A, but E was significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-1 was increased when HaCaT cells were cultured in a medium supplemented with an extract of Tamogitake acidic solvent, compared to the case where the cells were cultured without adding them.

  Further, as shown in the middle part of FIG. 5, regarding the relative expression level of hBD-2 mRNA, all of B, C, D, and E were almost the same as A.

  Further, as shown in the lower part of FIG. 5, regarding the relative expression level of hBD-3 mRNA, B and C were hardly changed compared to A, but D and E were significantly larger than A. . That is, it was confirmed that the mRNA expression level of hBD-3 increased when HaCaT cells were cultured in a medium supplemented with an extract of Tamogitake acidic solvent, compared with the case where the cells were cultured without adding them.

  From these results, it was clarified that Tamogitake acidic solvent extract promotes the production of hBD-1 and hBD-3 when allowed to act on HaCaT cells for 24 hours.

(5) Examination of human β-defensin production-promoting effect of Tamogitake basic solvent extract [5-1] Preparation of Tamogitake basic solvent extract Water was replaced with 1% (w / w) sodium bicarbonate aqueous solution. (3) An extract of Tamogitake was prepared by the method described in [3-1], and this was used as a Tamogitake basic solvent extract. The pH of the filtrate before sterilization with an autoclave was pH 7.6.

[5-2] Preparation and culture of sample and culture in medium supplemented with Tamogitake basic solvent extract A, B, C, D, and C by the method described in Example (1) [1-1] A total of 5 samples of E were prepared and cultured. Subsequently, instead of the Tamogitake hot water extract prepared in Example 1 (1), the Tamogitake basic solvent extract prepared in this Example (5) [5-1] was used, and this Example (1) was used. ) By the method as described in [1-2], it culture | cultivated with the culture medium which added the bamboo shoot basic solvent extract.

[5-3] Extraction of RNA, preparation of cDNA, and quantification of human β-defensin mRNA by quantitative PCR method For each sample of this example (5) [5-2], this example (1) [1- 3] After extracting RNA and preparing cDNA by the method described in [3], quantitative PCR is performed by the method described in Example (1) [1-4] to quantify human β-defensin mRNA. went. In addition, for the calculated results in B, C, D, and E, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 6, regarding the relative expression level of hBD-1 mRNA, B was almost the same as that of A, but C, D, and E were significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-1 was increased when HaCaT cells were cultured in a medium supplemented with the extract of Tamogitake basic solvent, compared with the case where the cells were cultured without adding them.

  Further, as shown in the middle part of FIG. 6, regarding the relative expression level of hBD-2 mRNA, all of B, C, D and E were almost the same as A.

  As shown in the lower part of FIG. 6, regarding the relative expression level of hBD-3 mRNA, all of B, C, and D were significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-3 was increased when HaCaT cells were cultured in a medium supplemented with an extract of Tamogitake basic solvent, compared to the case where the cells were cultured without adding them.

  From these results, it was clarified that Tamogitake basic solvent extract promotes the production of hBD-1 and hBD-3 when allowed to act on HaCaT cells for 24 hours.

(6) Examination of human β-defensin production promotion effect of ergothioneine derived from tamagotake mushroom [6-1] Preparation of ergothionein derived from tarogitake 150 kg to 200 kg of raw tarogitake was heated in 200 L of water and boiled for 5 minutes while boiling. Thereafter, filtration was performed to obtain 150 L of a filtrate. The filtrate was centrifuged at 5000 rpm and 10 ° C. for 30 minutes to recover 135 L of supernatant. 7.5 L of the collected supernatant was put into an ion exchange resin (Amberlite IR120B H type; Organo Co., Ltd.) packed in a 5 × 30 cm column and allowed to fall naturally overnight. Subsequently, the ion exchange resin is recovered, washed with 2.5 L of distilled water to remove saccharide components, and then eluted with 10 L of 0.28% (w / w) ammonia water. 10 mL of an eluate containing a cationic compound adsorbed on the solution was obtained. After concentrating the eluate using a rotary evaporator, high-performance liquid chromatography (HPLC) was performed according to a conventional method under the following conditions, and a peak fraction detected at 3 to 4 minutes from the start of effluent was collected.

HPLC Condition HPLC system; Hitachi High Performance Liquid Chromatograph LaChrom Elite
Elution solvent: 0 to 1% (v / v) acetonitrile aqueous solution column; Inertsil ODS-SP (GL Sciences Inc.)
Detector; UV detector detection condition; 250 nm

  The collected fractions were combined and then lyophilized using a freeze dryer to obtain 1.7 g (7.4 mmol) of powdered ergothioneine derived from Tamogitake.

[6-2] Preparation and culture of samples A total of 3 samples of A, B, and C were prepared and cultured by the method described in Example (1) [1-1].

[6-3] Cultivation in medium supplemented with bamboo shoot ergothioneine Example 6 (6) The bamboo shoot ergothioneine prepared in [6-1] was dissolved in DDW to give 10 mmol / L and 100 mmol / L ergothioneine, respectively. An aqueous solution was prepared. Next, DDW, 10 mmol / L and 500 mmol / L ergothioneine aqueous solutions were added to the FP-DMEM medium so as to be 1% (v / v), respectively. An exchange medium containing the concentration was prepared. After the medium of each sample of this example (6) [6-2] was replaced with the prepared replacement medium as described below, under the environment described in this example (1) [1-1], Cultured for 24 hours.

What was added to the exchange medium and final concentration A of ergothionein derived from Tamogitake A: DDW (exchange medium for control)
B: Tamogittake-derived ergothioneine final concentration 100 μmol / L
C: Tamogittake-derived ergothioneine final concentration 5 mmol / L

[6-4] Extraction of RNA, preparation of cDNA, and quantification of human β-defensin mRNA by quantitative PCR method For each sample of Example (6) [6-3], this Example (1) [1- 3] After extracting RNA and preparing cDNA by the method described in [3], quantitative PCR is performed by the method described in Example (1) [1-4] to quantify human β-defensin mRNA. went. In addition, Mann-Whitney U test was performed between the calculated results in B and C and the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 7, B and C were significantly larger than A in the relative expression level of hBD-1 mRNA. That is, it was confirmed that the mRNA expression level of hBD-1 was increased when HaCaT cells were cultured in a medium to which Tamogitake-derived ergothioneine was added, as compared to the case of culturing without adding it.

  Further, as shown in the middle part of FIG. 7, regarding the relative expression level of hBD-2 mRNA, both B and C were almost the same as A.

  In addition, as shown in the lower part of FIG. 7, regarding the relative expression level of hBD-3 mRNA, both B and C were significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-3 was increased when HaCaT cells were cultured in a medium supplemented with Tamogitake-derived ergothioneine as compared to the case where the cells were cultured without adding them.

  From these results, it was clarified that Tamogitake-derived ergothionein promotes the production of hBD-1 and hBD-3 when allowed to act on HaCaT cells for 24 hours.

(7) Examination of human β-defensin production-promoting effect of ergotosterol peroxide derived from tamagotake mushroom [7-1] Preparation of ergosterol peroxide derived from tarogitake 150 kg to 200 kg of raw tarogitake was heated in 200 L of water and boiled. After boiling for 5 minutes in the state, it was filtered to obtain a residue (residue). The filtrate was heated and dried, and then pulverized using a pulverizer until the particle diameter became 0.1 mm, to obtain 12 kg of powder. 99.5% (v / v) ethanol was added to 500 mL to 1000 mL per 100 g of the powder and heated to dissolve the components contained in the powder. After cooling this solution, it was centrifuged at 5000 rpm and 10 ° C. for 30 minutes to recover the supernatant, and an equal amount of 1 mol / L NaOH aqueous solution was added and stirred at room temperature for 2 hours. The reaction was carried out. Subsequently, the pH was adjusted to 5 to 6 by adding an appropriate amount of a 6 mol / L hydrochloric acid aqueous solution, followed by centrifugation at 5000 rpm and 10 ° C. for 30 minutes. After removing the supernatant, 100 mL of ethanol was added to the precipitate and stirred, and ethanol was distilled off under reduced pressure to obtain 10 to 50 g of a white precipitate, which was used as a crude lipid extract.

  Next, 10 to 50 g of the crude lipid extract was dissolved in 30 mL of chloroform / methanol {chloroform: methanol = 95: 5 (v / v)} and then packed with silica gel 60 (Kanto Chemical Co., Inc.) having a particle size of 100 to 210 μm. The solution was injected onto an 8 × 80 cm silica gel column. Subsequently, elution was performed with 1000 mL of chloroform / methanol {chloroform: methanol = 90: 10 (v / v)}, and 500 mL of the eluate was fractionated. An appropriate amount is collected from each fractionated eluate, and subjected to thin layer chromatography {developing solvent; chloroform: methanol: water = 80: 20: 0.2 (v / v)} according to a conventional method, and ergosterol peroxide is added. After selecting the eluate containing, the solvent of the selected eluate was distilled off under reduced pressure to obtain a crude purified product of ergosterol peroxide.

  Subsequently, the crude purified ergosterol peroxide was dissolved in chloroform / methanol {chloroform: methanol = 95: 5 (v / v)} and then packed with silica gel 60 (Kanto Chemical Co., Inc.) having a particle size of 40 to 100 μm. It inject | poured into the * 60cm silica gel column. Chloroform / methanol {chloroform: methanol = 95: 5 (v / v)} 1000 mL, chloroform / methanol {chloroform: methanol = 90: 10 (v / v)} 1000 mL, chloroform / methanol {chloroform: methanol = 85: 15 ( elution was performed with 1000 mL of v / v)} and 1000 mL of chloroform / methanol {chloroform: methanol = 80: 20 (v / v)}, and the eluate was fractionated by 18 to 50 mL. An appropriate amount is collected from each fractionated eluate, and subjected to thin layer chromatography {developing solvent; chloroform: methanol: water = 80: 20: 0.2 (v / v)} according to a conventional method, and ergosterol peroxide is added. After selecting the eluate to be contained, the solvent of the selected fraction was distilled off under reduced pressure to obtain 3 to 4 g (7.0 to 9.3 mmol) of purified Tamogittake-derived ergosterol peroxide.

[7-2] Sample Preparation and Culture A total of three samples A, B, and C were prepared and cultured by the method described in Example (1) [1-1].

[7-3] Cultivation in medium supplemented with bamboo shoot ergosterol peroxide In this example (7), taro shoot bamboo ergosterol peroxide prepared in [7-1] was dissolved in dimethyl sulfoxide, and each was 100 μmol / L and 1 mmol / L ergosterol peroxide aqueous solutions were prepared. Next, dimethyl sulfoxide, 100 μmol / L and 1 mmol / L ergosterol peroxide aqueous solution were added to the FP-DMEM medium so as to be 1% (v / v), respectively. An exchange medium containing sterol peroxide at the following final concentration was prepared. After the medium of each sample of this example (7) [7-2] was replaced with the prepared replacement medium as described below, under the environment described in this example (1) [1-1], Cultured for 24 hours.

What was added to the exchange medium and the final concentration A of ergosterol peroxide derived from Tamogitake A: dimethyl sulfoxide (exchange medium for control)
B: Tamogittake-derived ergosterol peroxide final concentration 1 μmol / L
C: Tamogitake-derived ergosterol peroxide final concentration 10 μmol / L

[7-4] Extraction of RNA, preparation of cDNA, and quantification of human β-defensin mRNA by quantitative PCR method For each sample of this example (7) [7-3], this example (1) [1- 3] After extracting RNA and preparing cDNA by the method described in [3], quantitative PCR is performed by the method described in Example (1) [1-4] to quantify human β-defensin mRNA. Went. In addition, Mann-Whitney U test was performed between the calculated results in B and C and the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 8, regarding the relative expression level of hBD-1 mRNA, both B and C were almost the same as A.

  Further, as shown in the middle part of FIG. 8, regarding the relative expression level of hBD-2 mRNA, both B and C were significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-2 was increased when HaCaT cells were cultured in a medium supplemented with tamagotake-derived ergosterol peroxide as compared to the case where the cells were cultured without adding them.

  Further, as shown in the lower part of FIG. 8, regarding the relative expression level of hBD-3 mRNA, C tends to be larger than A, and B is significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-3 was increased when HaCaT cells were cultured in a medium supplemented with tamagotake-derived ergosterol peroxide as compared to the case where the cells were cultured without adding them.

  From these results, it has been clarified that ergotosterol peroxide derived from Tamogitake promotes the production of hBD-2 and hBD-3 when allowed to act on HaCaT cells for 24 hours.

(8) Examination of human β-defensin production-promoting effect of glucosylceramide derived from Tamogitake [8-1] Preparation of glucosylceramide derived from Tamogitake For each eluate from the silica gel column, the contained components are confirmed and selected by thin layer chromatography. In this case, instead of ergosterol peroxide, an eluate containing glucosylceramide is selected, and the method described in Example (7) [7-1] is performed, so that the purified Tamimogi-derived glucosylceramide 20 to 25 g (0.028-0.034 mol) were obtained.

[8-2] Preparation and Culture of Samples A total of 5 samples A, B, C, D and E were prepared and cultured by the method described in Example (1) [1-1].

[8-3] Cultivation in medium supplemented with bamboo shoot-derived glucosylceramide The bamboo shoot-derived glucosylceramide prepared in Example (8) [8-1] was dissolved in dimethyl sulfoxide, and each was 100 μmol / L, 1 mmol / L, 10 mmol / L and 100 mmol / L glucosylceramide aqueous solutions were prepared. Next, dimethyl sulfoxide, 100 μmol / L, 1 mmol / L, 10 mmol / L, and 100 mmol / L glucosylceramide aqueous solution were added to the FP-DMEM medium to be 0.1% (v / v), respectively. An exchange medium for control and an exchange medium containing Tamogitake-derived glucosylceramide at the following final concentrations were prepared. After the medium of each sample of this example (8) [8-2] was replaced with the prepared replacement medium as described below, under the environment described in this example (1) [1-1], Cultured for 24 hours.

What was added to the exchanged medium and the final concentration of glucosylceramide derived from Tamogitake A: dimethyl sulfoxide (exchange medium for control)
B: Glucosylceramide derived from Tamogitake final concentration 0.1 μmol / L
C: Tamogittake-derived glucosylceramide final concentration 1 μmol / L
D: Tamogitake glucosylceramide final concentration 10 μmol / L
E: Glucosylceramide derived from Tamogitake final concentration of 100 μmol / L

[8-4] Extraction of RNA, preparation of cDNA, and quantification of human β-defensin mRNA by quantitative PCR method For each sample of this example (8) [8-2], this example (1) [1- 3] After extracting RNA and preparing cDNA by the method described in [3], quantitative PCR is performed by the method described in Example (1) [1-4] to quantify human β-defensin mRNA. Went. In addition, for the calculated results in B, C, D, and E, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 9, regarding the relative expression level of hBD-1 mRNA, B, C and D showed no significant difference compared to A, and the relative expression level of mRNA in E was the relative expression level of mRNA in A. It was significantly smaller than the amount.

  In addition, as shown in the middle part of FIG. 9, regarding the relative expression level of hBD-2 mRNA, no significant difference was observed in all of B, C, D and E as compared with A.

  In addition, as shown in the lower part of FIG. 9, regarding the relative expression level of hBD-3 mRNA, B hardly changed compared to A, but C and D tended to be large compared to A, and E was It was significantly larger than A. That is, it was confirmed that the mRNA expression level of hBD-3 was increased when HaCaT cells were cultured in a medium supplemented with Tamogitake-derived glucosylceramide as compared to when cultured without adding it.

  From these results, it was clarified that the glucosylceramide derived from Tamogitake promotes the production of hBD-3 when allowed to act on HaCaT cells for 24 hours.

<Example 3> Examination of human β-defensin production promoting effect in NHEK cells (1) Preparation and culture of sample According to the method described in Example 2 (1) [1-1], A, B, C and D A total of 4 samples were prepared. However, normal human epidermal keratinocytes (NHEK cells; Sanko Junyaku Co., Ltd.) were used instead of HaCaT cells, and the culture time was 2 days instead of 3 days.

(2) Culture in medium supplemented with Tamogitake hot water extract For each sample of this Example (1), Tamogittake hot water extract was added by the method described in Example 2 (1) [1-2]. Culturing was performed on the prepared medium. However, the exchange medium used for each sample was as follows.

What was added to the exchange medium and the final concentration A of hot water extract of Tamogitake A: DDW (exchange medium for control)
B: Tamogitake hot water extract final concentration 0.5% (v / v)
C: Tamogitake hot water extract final concentration 5% (v / v)
D: Tamogitake hot water extract final concentration 25% (v / v)

(3) Extraction of RNA, preparation of cDNA, and quantification of hBD-2 mRNA and hBD-3 mRNA by quantitative PCR method About each sample of this Example (2), it is described in Example 2 (1) [1-3]. After extracting RNA and preparing cDNA by the method, quantitative PCR was performed by the method described in Example 2 (1) [1-4] to quantify hBD-2 mRNA and hBD-3 mRNA. In addition, for the calculated results in B, C, and D, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 10, regarding the relative expression level of hBD-2 mRNA, B was hardly changed compared to A, but C and D were significantly larger than A.

  In addition, as shown in the lower part of FIG. 10, regarding the relative expression level of hBD-3 mRNA, B was hardly changed compared to A, but C and D were significantly larger than A. That is, it is confirmed that the mRNA expression level of hBD-2 and hBD-3 increases when NHEK cells are cultured in a medium supplemented with Tamogitake hot-water extract, compared with the case where the cells are cultured without addition of NHEK cells. It was done.

  From these results, it has been clarified that the Tamogitake hot water extract promotes the production of hBD-2 and hBD-3 not only in HaCaT cells but also in NHEK cells.

<Example 4> Examination of time to exert human β-defensin production promoting effect (1) Tamogitake hot water extract Example 2 (1) Experiments were performed by the method described in [1-1] to [1-4]. went. However, the culture time in the medium supplemented with Tamogitake hot water extract was 2 hours instead of 24 hours, and the quantification of human β-defensin mRNA by quantitative PCR was performed for hBD-2 and hBD-3. In addition, for the calculated results in B, C, D, and E, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 11, regarding the mRNA relative expression level of hBD-2, B and C were hardly changed compared to A, but D and E were significantly larger than A.

  In addition, as shown in the lower part of FIG. 11, regarding the relative expression level of hBD-3 mRNA, B was hardly changed compared to A, but C, D, and E were significantly larger than A.

  From these results, it is clear that the Tamogitake hot water extract promotes the production of hBD-2 and hBD-3 when it is allowed to act on HaCaT cells for 2 hours, similarly to the case where it is allowed to act for 24 hours. It was. In addition, it has been clarified that the degree of promotion of the production of hBD-2 and hBD-3 by the Tamogitake hot water extract is greater when it is allowed to act for 2 hours than when it is allowed to act for 24 hours. .

(2) Filtrate and filtrate of Tamogitake hot water extract An experiment was conducted by the method described in Example 2 (2) [2-1] to [2-4]. However, the culture time in the culture medium to which the filtrate of the water extract of Tamogitake and the filtrate was added was set to 2 hours instead of 24 hours. In addition, for the calculated results in B, C, and D, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in FIG. 12, regarding the relative expression level of hBD-3 mRNA, B tended to be larger than A, and C and D were significantly larger than A.

  From these results, it is clarified that the filtrate of Tamogitake hot water extract tends to promote the production of hBD-3 when it is allowed to act on HaCaT cells for 2 hours, similarly to the case where it is allowed to act for 24 hours. It was. In addition, it has been clarified that the filtrate obtained from the extract of hot water extract of Tamogitake promotes the production of hBD-3 when it is allowed to act on HaCaT cells for 2 hours, similarly to the case where it is allowed to act for 24 hours. In addition, the degree to which the filtrate and filtrate of Tamogitake hot water extract promote the production of hBD-3 is slightly greater when operated for 24 hours than when operated for 24 hours. It was.

(3) Ultrasonic treatment product of Tamogitake hot water extract [3-1] Preparation of ultrasonic treatment product of Tamogitake hot water extract 20 mL of Tamogitake hot water extract prepared in Example 1 (1) Then, using an ultrasonic generator, ultrasonic waves of 40 kHz were irradiated for 50 minutes to obtain an ultrasonically treated product of the water extract of Tamogitake.

[3-2] Preparation and culture of samples A total of 3 samples of A, B, and C were prepared and cultured by the method described in Example 2 (1) [1-1].

[3-3] Culture of Tamogitake hot water extract in medium supplemented with sonicated product Diluted sonicated product of Tamogitake hot water extract of Example (3) [3-1] using DDW Then, a 10% (v / v) sonicated aqueous solution was prepared. Next, 10% (v / v) each of the ultrasonically treated product of DDW, 10% (v / v) sonicated product aqueous solution and the Tamogitake hot water extract of Example (3) [3-1] of this example. ) To the FP-DMEM medium to prepare a control exchange medium and an exchange medium containing the sonicated product of the Tamogitake hot water extract at the following final concentrations. The medium of Example (3) [3-2] was replaced with the prepared replacement medium as described below, and then further cultured for 2 hours in the environment described in Example 2 (1) [1-1]. did.

The final concentration A of the sonicated product of the one added to the exchange medium and the Tamogitake hot water extract A: DDW (exchange medium for control)
B: Ultrasonic processed product of Tamogitake hot water extract Final concentration 1% (v / v)
C: Ultrasonic treatment product of Tamogitake hot water extract 10% final concentration (v / v)

[3-4] Extraction of RNA, preparation of cDNA, and quantification of hBD-3 mRNA by quantitative PCR method For each sample of Example (3) [3-3], Example 2 (1) [1-3] After extracting RNA and preparing cDNA by the method described in 1., quantitative PCR was performed by the method described in Example 2 (1) [1-4] to quantify hBD-3 mRNA. In addition, Mann-Whitney U test was performed between the calculated results in B and C and the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in FIG. 13, regarding the relative expression level of hBD-3 mRNA, B and C were both larger than A, and the order of the relative expression level of mRNA was A <B <C.

  From these results, it was clarified that the sonicated product of the extract of hot water from Tamogitake promotes the production of hBD-3 when allowed to act on HaCaT cells for 2 hours.

(4) Tamogitake water extract An experiment was conducted by the method described in Example 2 (3) [3-1] to [3-3]. However, the culture time in the medium supplemented with the water extract of Tamogitake was 2 hours instead of 24 hours. In addition, for the calculated results in B, C, D, and E, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 14, regarding hBD-1 mRNA relative expression level, B and D were almost the same as A, but C and E were significantly larger than A.

  Further, as shown in the middle part of FIG. 14, regarding the relative expression level of hBD-2 mRNA, all of B, C, D and E were almost the same as A.

  In addition, as shown in the lower part of FIG. 14, regarding the relative expression level of hBD-3 mRNA, B and C were hardly changed compared to A, but C and E were significantly larger than A. .

  From these results, it was clarified that the water extract of Tamogitake promotes the production of hBD-1 and hBD-3 when it was allowed to act on HaCaT cells for 2 hours, as was the case when it was allowed to act for 24 hours. . In addition, it has been clarified that the degree of promotion of the production of hBD-1 and hBD-3 by the water extract of Tamogitake is greater when it is allowed to act for 24 hours than when it is allowed to act for 2 hours.

(5) Tamogitake acidic solvent extract An experiment was conducted by the method described in Example 2 (4) [4-1] to [4-3]. However, the culturing time in the medium supplemented with the extract of the acidic solvent extract was 2 hours instead of 24 hours. In addition, for the calculated results in B, C, D, and E, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 15, regarding the mRNA relative expression level of hBD-1, B and C were almost the same as A, but D and E were significantly larger than A.

  In addition, as shown in the middle part of FIG. 15, regarding the relative expression level of hBD-2 mRNA, all of B, C, D and E were almost the same as A.

  Further, as shown in the lower part of FIG. 15, regarding the relative expression level of hBD-3 mRNA, C and E were almost the same as A, but B and D were significantly larger than A. .

  From these results, it is clarified that Tamogitake acidic solvent extract promotes the production of hBD-1 and hBD-3 when it is allowed to act on HaCaT cells for 2 hours, similarly to the case where it is allowed to act for 24 hours. It was. In addition, the extent to which Tamogitake acidic solvent extract promotes the production of hBD-1 is the same as the case of acting for 2 hours and the case of acting for 24 hours, and the extent of promoting the production of hBD-3 is It became clear that the case of 24 hours of action was greater than the case of 2 hours of action.

(6) Tamogitake basic solvent extract An experiment was conducted by the method described in Example 2 (5) [5-1] to [5-3]. However, the culture time in the medium supplemented with Tamogitake basic solvent extract was 2 hours instead of 24 hours. In addition, for the calculated results in B, C, D, and E, Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 16, B, C, D, and E were significantly larger than A in the hBD-1 mRNA relative expression level.

  Further, as shown in the middle part of FIG. 16, regarding the mRNA relative expression level of hBD-2, all of B, C, D and E were almost the same as A.

  In addition, as shown in the lower part of FIG. 16, regarding the mRNA relative expression level of hBD-3, B and D were hardly changed compared to A, but C and E were significantly larger than A. .

  From these results, it is clear that Tamogitake basic solvent extract promotes the production of hBD-1 and hBD-3 when it is allowed to act on HaCaT cells for 2 hours, similarly to the case where it is allowed to act for 24 hours. became. In addition, it has been clarified that the extent of the Tamogitake basic solvent extract promoting the production of hBD-1 and hBD-3 is almost the same between the case of acting for 2 hours and the case of acting for 24 hours.

(7) Tamogittake-derived ergothioneine An experiment was conducted by the method described in Example 2 (6) [6-1] to [6-4]. However, the culture time in the medium supplemented with Tamogitake ergothionein was 2 hours instead of 24 hours. In addition, Mann-Whitney U test was performed between the calculated results in B and C and the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper, middle and lower parts of FIG. 17, B and C were significantly larger than A in all of the relative mRNA expression levels of hBD-1, hBD-2 and hBD-3.

  From these results, ergothioneine derived from Tamogitake not only promotes the production of hBD-1 and hBD-3, but also acts for 24 hours when acted on HaCaT cells for 2 hours. It was revealed that the production of hBD-2 that was not promoted was also promoted. In addition, it has been clarified that the degree of promotion of the production of hBD-1 and hBD-3 by Tamogitake-derived ergothionein is slightly greater when it is allowed to act for 24 hours than when it is allowed to act for 2 hours.

<Example 5> Examination of human β-defensin production-promoting effect in the case where a Tamogitake hot water extract is added together with β-glucan or mycoplasma-derived diacyl lipopeptide (FSL-1) (1) Sample preparation and culture Example 2 ( 1) A total of 8 samples of A, B, C, D, E, F, G and H were prepared by the method described in [1-1]. However, NHEK cells (Sanko Junyaku Co., Ltd.) were used instead of HaCaT cells, and the culture time was 2 days instead of 3 days.

(2) Culture in medium supplemented with β-glucan, FSL-1 and Tamogitake hot water extract Suehiro bamboo-derived β-glucan (Soniphylan; Kaken Pharmaceutical), barley-derived β-glucan (MP Biomedicals), Toll Like Receptor (TLR) -2 and TLR-6 agonist Mycoplasma-derived diacyl lipopeptide (FSL-1; InvivoGen) and Tamogitake hot water extract prepared in Example 1 (1) were diluted with DDW or dissolved in DDW The following aqueous solutions of b, c, d, e and g were prepared. In addition, the following f and h Tamogitake hot water extract solutions were prepared by dissolving Shirohirotake-derived β-glucan or FSL-1 in the Tamogitake hot water extract.

Concentration b of aqueous solution and Tamogittake hot water extract solution: β-glucan derived from Suehirotake 100 μg / mL
c: Barley-derived β-glucan 100 μg / mL
d: FSL-1 100 ng / mL
e: Tamogitake hot water extract 50% (v / v)
f: Tamogitake hot water extract 100% (v / v) + Suehirotake-derived β-glucan 200 μg / mL
g: FSL-1 200 μg / mL + Suehirotake-derived β-glucan 200 μg / mL
h: Tamogitake hot water extract 100% (v / v) + FSL-1 200 μg / mL

  Next, DDW, each prepared aqueous solution and each Tamogitake hot water extract solution were respectively added to FP-DMEM medium so as to be 5% (v / v), and Suehirotake-derived β-glucan (Sonifilan; Kaken Pharmaceutical Co., Ltd.) ), Barley-derived β-glucan (MP Biomedicals), FSL-1 (InvivoGen), and Tamogitake hot water extract at the following final concentrations and an exchange medium for control were prepared. The medium of this example (1) was replaced with the prepared exchange medium as described below, and then further cultured for 2 hours in the environment described in Example 2 (1) [1-1].

What was added to the exchange medium and its final concentration A: DDW (exchange medium for control)
B: Suehirotake-derived β-glucan final concentration 10 μg / mL
C: Barley-derived β-glucan final concentration of 10 μg / mL
D: FSL-1 final concentration 10 ng / mL
E: Tamogitake hot water extract final concentration 5% (v / v)
F: Tamogitake hot water extract final concentration 5% (v / v) + Suehirotake-derived β-glucan final concentration 10 μg / mL
G: FSL-1 final concentration 10 ng / mL + Suehirotake-derived β-glucan final concentration 10 μg / mL
H: Tamogitake hot water extract final concentration 5% (v / v) + FSL-1 final concentration 10 ng / mL

(3) Extraction of RNA, preparation of cDNA, and quantification of hBD-2 mRNA and hBD-3 mRNA by quantitative PCR method About each sample of this Example (2), it is described in Example 2 (1) [1-3]. After extracting RNA and preparing cDNA by the method, quantitative PCR was performed by the method described in Example 2 (1) [1-4] to quantify hBD-2 mRNA and hBD-3 mRNA. In addition, for the calculated results in B, C, D, E, F, G, and H, the Mann-Whitney U test was performed with the results in A. The result is shown in FIG. * In the figure indicates that the result of the U test is statistically significant (p <0.05).

  As shown in the upper part of FIG. 18, regarding the relative expression level of hBD-2 mRNA, all of B, C, D, E and H are slightly larger than A, and F and G are significantly larger than A. It was big.

  Further, as shown in the lower part of FIG. 18, as for hBD-3 mRNA relative expression level, as with hBD-2, all of B, C, D, and E are slightly larger than A, and F and G are It was significantly larger than A.

  From these results, it was clarified that the effect of accelerating the production of hBD-2 and hBD-3 is improved by allowing the Tamogitake hot water extract to act together with β-glucan.

Example 6 Confirmation of Antibacterial Effect in Oral Candidiasis Model Mice (1) Preparation of Oral Gel Containing Tamogitake Hot Water Extract Tamogitake Hot Water Extract and White Petrolatum (Yakuhan) Prepared in Example 1 (1) Was added to a dental dappen glass, and stirred and mixed using a plastic cement spatula to prepare an oral gel containing 60% (w / w) of a Tamogitake hot water extract.

(2) Production of oral candidiasis model mice
Oral candidiasis model mice were prepared according to the previous report (Takakura et al., Microbiol. Immunol., 47, No. 5, 321-326, 2003). Specifically, 15 6-week-old C3H / HeJJcl male mice (CLEA Japan, Inc.) were subcutaneously injected with 100 mg of prednisolone per kg body weight of the mouse. Subsequently, sterilized tap water added with chlortetracycline hydrochloride to 0.83 mg / mL was bred for 24 hours while freely ingesting as drinking water, and then 2 mg / mL chlorpromazine hydrochloride aqueous solution per mouse. 50 mL was intramuscularly injected into the thigh. After 30 minutes, a suspension of Candida albicans (Candida albicans ATCC 90028) suspension at 1 × 10 ⁸ cfu / mL was attached to a sterile cotton swab, and this was used to inoculate Candida bacteria into the tongue and oral cavity of mice.

(3) Administration of oral gel containing Tamogitake hot water extract 15 mice of this Example (2) are divided into 7 and 8 mice, a group consisting of 7 mice is a control group, a group consisting of 8 mice As a gel administration group, each group of mice was bred for 3 days. During this time, 0.05 g per mouth of the oral gel of this Example (1) was applied three times a day only to the gel administration group.

(4) Measurement of the number of Candida bacteria The tongue was extracted from each group of mice of Example (3), and 1 mL of physiological saline was added per 100 mg of tongue weight to obtain a suspension. About 100 μL of this suspension, the number of Candida viable cells was measured by performing a plate method according to a standard method using CHIROMaga selective medium (CHIROMagar), and the average value and the standard deviation of the number of viable cells were determined for each group. . Further, the Man Whitney U test was performed between the calculated results in the gel administration group and the results in the control group. The result is shown in FIG. * In the figure indicates that the result of the test is statistically significant (p <0.05).

  As shown in FIG. 19, the viable count of Candida in the gel administration group is significantly smaller than the viable count of Candida in the control group, and the viable count of Candida in the control group is 100%. The viable count of Candida in the gel administration group was 77.5%. In other words, it was confirmed that the oral gel containing the Tamimogi hot water extract suppresses the growth of Candida bacteria in the oral cavity of oral candidiasis model mice.

  From this result, it was revealed that the oral gel containing the Tamimogi hot water extract is effective for the prevention or treatment of oral candidiasis.

Claims (3)

An anti- Candida fungus containing hot water extract of Tamogitake as an active ingredient.   A preventive and / or therapeutic agent for candidiasis comprising a hot water extract of Tamogitake as an active ingredient.   The agent for preventing and / or treating candidiasis according to claim 2, which is used for a patient with xerostomia.

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